There are three main strategies for using oligonucleotides to affect gene regulation. These strategies involve the use of antisense oligonucleotides, triplex (or antigene) oligonucleotides, and ribozymes. Each strategy has its inherent advantages and limitations, and each has a growing base of experimental successes.
Antisense Oligonucleotides
Antisense oligonucleotides are the earliest examples of oligonucleotide-based approaches to gene regulation. Not only were they the first to be tested for biological activity in the laboratory and the clinic, but also the antisense concept may have first arisen in prokaryotes. Naturally occurring antisense RNA has been detected in E. coli bacteria as well as in colE1 and IS10 plasmids. In these strains, regions of transcribed RNA in the antisense orientation serve to regulate the translation of RNA in the sense orientation (Inouye, M. (1988) Gene 72:25-34; and Simons, R. W. and Kleckner, N. (1988) Annual Rev. Gen. 22:567-600). Similar naturally occurring antisense regulation strategies have now been identified in several eukaryotic genes as well (Bentley, D. L. and Groudine, M. (1986) Nature 321:702-706; Kimelman, D. Gene regulation: Biology of Antisense RNA and DNA, R. P. Erickson, J. G. Izant, eds. (Raven Press, New York) pp. 1-10). Given that the normal messenger RNA (mRNA) transcribed from DNA is referred to as the "sense" RNA strand, oppositely oriented RNA are termed antisense RNA. Antisense oligonucleotides, then, refer to specific sequences of DNA or RNA which can bind in a Watson-Crick fashion to a sequence on a target mRNA.
In forming a double-stranded region on the mRNA, subsequent steps of protein synthesis may be interrupted by any of a variety of mechanisms. Interruption may occur by sterically blocking ribosome assembly or progression, sterically blocking intron/exon junctions and splice-sites needed for the processing of premature mRNA, or by invoking the cellular enzyme RNAse H that specifically cleaves mRNA in mRNA/DNA hybrids. The potential of antisense oligonucleotides to enact specific inhibition of protein synthesis is reflected in the tremendous number of publications which have appeared in the nearly 20 years since the earliest report of an antisense effect against the Rous Sarcoma Virus (Zamecnik, P. C. and Stephenson, M. L. (1978) Proc. Natl. Acad. Sci. USA 75:280-284; and for general reference on antisense oligonucleotides see: Moffat, A. S. (1991) Science 253:510-511; Chrisey, L. A. and Hawkins, J. W. (1991) Biopharm. 36-42; Oligonucleotides: Antisense Inhibitors of Gene Expression (1989) J. S. Cohen, ed. (MacMillan Press, London); and Stein, C. A. and Cheng, Y. C. (1993) Science 261:1004-1012). Antisense RNA and DNA has been demonstrated to lead as much as 95% inhibition of specific mRNA translation. In some cases, the antisense strategy has led to upregulated levels of the corresponding protein (Williard, R. L. et al. (1994) Gene (in press)).
A single gene encoded in DNA can, and most often will, be transcribed multiple times, giving rise to many copies of mRNA. In turn, a single mRNA molecule can, and most often will, be translated multiple times giving rise to many copies of the corresponding protein. Therefore, amplification can take place both at the DNA-&gt;mRNA level, and also at the mRNA-&gt;protein level. Antisense oligonucleotides can be very effective at blocking the translation of RNA and reducing the amount of protein synthesized, but they must be present within the cell in sufficient numbers to account for the previous DNA-&gt;mRNA amplification step. If a cell contains a compensatory response to lower levels of a given protein, feedback may upregulate the transcription of the corresponding RNA, increase mRNA stability, or signal for an increase in ribosomal assembly--all of which may serve to diminish the efficacy of an antisense approach. Given this constraint, it is unlikely that 100% inhibition of target gene expression could be achieved. To achieve this maximal level of inhibition, one must backstep and posit a strategy in which mRNA transcription (and thus the first level of amplification) is prevented. Such a strategy can be theoretically achieved by triple helix-forming (triplex) oligonucleotides.
Triplex Oligonucleotides
The development of triplex oligonucleotides as inhibitors of gene expression is receiving considerable, albeit delayed, attention. In 1957, Felsenfeld, Davies, and Rich described a surprisingly stable structure composed of three homopolymeric nucleic acid strands (Felsenfeld, G. et al. (1957) J. Am. Chem. Soc. 79:2023-2024). Two of these strands formed normal Watson-Crick hydrogen bonds, while the third strand was associated by what are now called Hoogsteen hydrogen bonds. Later studies confirmed and elaborated upon these findings, and determined that a pyrimidine-rich third strand could reside in the major groove of homopurine/homopyrimidine double-stranded DNA or RNA without perturbation of underlying Watson-Crick base-pairs. The binding was found to be pH-dependent (when cytosine residues were involved), and oriented parallel to the corresponding purine strand of the double-stranded helix (Lyamichev, V. I. et al. (1986) J. Biomol. Struct. and Dynam. 3:667-669; and Moser, H. E. and Dervan, P. B. (1987) Science 238:645-650). Nearly three decades passed before several laboratories began to use triplex oligonucleotides both to create a new class of sequence-specific DNA cleaving tools (Moser, H. E. and Dervan, P. B. (1987) Science 238:645-650; Strobel, S. A. and Dervan, P. B. (1990) Science 249:73-73; Strobel, S. A. and Dervan, P. B. (1991) Nature 350:172-174; and Dervan, P. B. Oligonucleotides: Antisense Inhibitors of Gene Expression, J. S. Cohen, ed. (Macmillan Press, London) pp. 197-210) as well as to mediate specific gene regulation at the level of the promoter (Durland, R. H. et al. (1991) Biochem. 30:9246-9255; Duval-Valentin, G. et al. (1992) Proc. Natl. Acad. Sci. USA 89:504-508; and Maher, L. J. et al. (1989) Science 245:725-730).
Subsequently, a number of reports emerged citing the ability of a sequence-specific pyrimidine-rich oligonucleotide to inhibit the binding of DNA binding proteins and in vitro gene transcription and elongation (Maher, L. J. et al. (1992) Biochem. 31:70-81; Young SL et al. (1991) Proc. Natl. Acad. Sci. USA 88:10023-10026; and Cooney, M. et al. (1988) Science 241:456-459). In addition to the pyrimidine-rich third strand binding motif previously described, a second triplex binding motif was identified involving a purine-rich third strand bound in a Mg++-dependent, pH-independent fashion (Chamberlin, M. J. and Patterson D. L. (1965) J. Mol. Biol. 12:410-428; Cooney, M. et al. (1988) Science 241:456-459; and Beal, P. A. and Dervan, P. B. (1991) Science 251:1360-1363). Orientation was shown to be antiparallel when the third strand was composed of G and A residues and dependent upon GpA steps in the target sequence when the third strand was composed of G and T residues (Sun, J. S. et al. (1991) C. R. Acad. Sci. Paris Serial III 313:585-590). A growing number of reports of in vivo inhibition of gene expression have been cited using triplex oligonucleotides of this second binding motif, while only triplex oligonucleotides conjugated to crosslinking agents have shown similar capabilities when using the first binding motif (Postel, E. H. et al. (1991) Proc. Natl. Acad. Sci. USA 88:8227-8231; McShan, W. M. et al. (1992) J. Biol. Chem. 267:5712-5721; Ing, N. H. et al. (1993) Nuc. Acids Res. 21:2789-2796; Roy, C. (1993) Nuc. Acids Res. 21:2845-2852); and Grigoriev, M. et al. (1993) Proc. Natl. Acad. Sci. USA 90:3501-3505).
While the majority of reports which cite triplex-based repression of transcription both in vitro and in vivo have utilized DNA triplex oligonucleotides on DNA double-stranded targets, recent research indicates that the thermodynamics of binding may favor an RNA third strand over a DNA third strand should a pyrimidine-rich binding motif be chosen (Roberts, R. W. and Crothers, D. M. (1992) Science 258:1463-1467; and Escude, C. et al. (1993) Nuc. Acids Res. 21:5547-5553). However, RNA purine-rich oligonucleotides have not been shown to form triplex structures with DNA double-stranded targets under any known conditions (Escude, C. et al. (1993) Nuc. Acids Res. 21:5547-5553; and Noonberg, S. B. et al. (1994) BioTechniques 16:1070-1073), but have been shown to form triplex structures with RNA homopurine/hompyrimidine duplexes and DNA/RNA hybrid duplexes (Chamberlin, M. J. and Patterson, D. L. (1965) J. Mol. Biol. 12:410-428). Such results underscore the importance of differing sugar backbones on triplex formation and stability.
A great deal of research effort and resources are being devoted to triplex oligonucleotides due to their potential to abolish completely the expression of a specific protein, especially when conjugated to intercalating agents which can crosslink the underlying DNA at a given triplex site (Grigoriev, M. et al. (1993) Proc. Natl. Acad. Sci. USA 90:3501-3505; and Helene, C. and Toulme, J. J. in Oligonucleotides: Antisense Inhibitors of Gene Expression, J. S. Cohen, ed. (Macmillan Press, London) pp. 137-172). Whether the triplex structure is permanent or in a binding equilibrium, triplex formation may inhibit the expression of a given gene in a variety of mechanisms: steric interference with transcription factor binding and assembly on promoter targets, alteration of duplex rigidity and inability for nonadjacent DNA binding proteins to associate, alteration of major and minor grooves to prevent neighboring transcription factor binding, and inhibition of elongation of the transcription complex producing truncated mRNA transcripts. In addition, a covalent complex may induce DNA repair elements to cleave out the linkage and thus inactivate a regulatory region of a gene.
Ribozymes
First identified within the RNA of Tetrahymenae (Kruger, K. (1982) Cell 31:147-157), ribozymes are able to perform self-cleavage reactions without additional protein enzyme or catalytic elements. This feature suggests that ribozymes may represent the first self-replicating entity. Ribozymes have now been isolated (primarily from viral sources) in various species of plants, fungi, and animals, all sharing the same capacity for catalytic activity without additional protein co-factors (Foster, A. C. and Symons, R. H. (1987) Cell 49:211-220). Their possible role in selective gene regulation stems from the ability to couple the catalytic RNA center domain to flanking RNA sequences designed to target an mRNA molecule by Watson-Crick base-pairing. Thus, the ribozyme can bind and cleave a target mRNA without self-impairment. Like antisense oligonucleotides, ribozymes also act on mRNA as opposed to DNA, and therefore do not affect the initial amplification step of transcription. And like antisense oligonucleotides, it may be near impossible to achieve 100% inhibition of target gene expression using a ribozyme strategy. However, unlike antisense oligonucleotides, ribozymes have the potential to quickly, permanently, and repetitively inactivate substrate mRNA. These differences may allow for the detection of significant biological activity at far lower concentrations intracellularly.
As with antisense and triplex oligonucleotides, a growing number of reports of ribozyme-mediated suppression of protein synthesis are appearing (Cotten, M. and Birnstiel, M. L.(1989) EMBO J. 8:3861-3868; Sarver, M. et al. (1990) Science 247:1222-1224; Cameron, F. H. and Jennings, P. A. (1989) Proc Natl Acad Sci USA 86:9139-9143; and Haseloff, J. and Gerlach, W. L. Nature 334: 585-591).
Increasing attention has been drawn to employing antisense, ribozyme, and triplex forming oligonucleotides in strategies for specific gene regulation. Oligonucleotides may specifically bind to viral DNA and RNA sequences involved in viral replication and pathogenicity, cellular oncogenic DNA and RNA sequences involved in neoplastic cell proliferation and differentiation, and various protein coding DNA and RNA sequences involved in disease pathophysiology. Oligonucleotides are thought to have potential utility in antiviral, anticancer, and antiprotein therapeutics.
Historically, oligonucleotides have been introduced into cells in one of three ways: by addition to the extracellular media, by invasive techniques such as electroporation or microinjection, or by integration of antisense mRNA vectors into the host chromosome. Each of these methods has its advantages and limitations. The first method, the addition of oligonucleotides to the media bathing the cells, induces little cellular damage and delivers short nucleic acid sequences, but the mechanisms of oligonucleotide uptake have poor efficiency and the cost of oligonucleotide synthesis is significant.
The second class of techniques, microinjection and electroporation, can greatly increase the efficiency of uptake of small oligonucleotides into the cell, but may cause significant cellular injury and disruption of normal cellular function. The third class of techniques, integration of the target gene and RNA polymerase II promotor in an antisense orientation via plasmids or retroviral vectors, can provide stable transcripts in healthy cells, but these transcripts retain their full length and can be expected to display considerable secondary structure, masking key antisense regions. In addition, RNA polymerase II transcripts are not continually produced, and transcriptional frequency is quite low in comparison to RNA products from polymerases I and III.
Interest in the biological activity of triplex-forming oligonucleotides has been steadily increasing, owing in part to their potential as artificial repressors of gene expression (Helene, C. (1991) Anticancer Drug Design 6:569-584; Maher III, L. J. et al. (1989) Science 245:725-730; Postel, E. H. et al. (1991) Proc. Natl. Acad. Sci. USA 88:8227-8231; and Ing, N. H. et al. (1993) Nucleic Acids Res. 21:2789-2796) as well as mediators of site-specific DNA cleavage (Moser, H. E. and Dervan, P. B. (1987) Science 238:645-650; and Strobel, S. A. and Dervan, P. B. (1991) Nature 350:172-174).
The potential of triplex and antisense oligonucleotides to inhibit selectively protein synthesis from a specified target gene has generated significant enthusiasm for their development as experimental therapeutics. Inhibition of expression of virally-derived proteins (Zamecnik & Stephenson (1978) Proc. Natl. Acad. Sci. USA 75: 280-284; Agrawal, S. (1991) In Prospects for Antisense Nucleic Acid Therapy of Cancer and AIDS. Eric Wickstrom, ed. (Wiley-Liss, New York), pp. 143-158; and McShan, W. M. et al. (1992) J. Biol. Chem. 267: 5712-5721) or endogenously activated oncogenes that contribute to cancer induction and/or progression (Helene, C. (1991) Anticancer Drug Design 6: 569-584; Postel, E. H. et al. (1991) Proc. Natl. Acad. Sci. USA 88: 8227-8231; and Calabretta, B. (1991) Cancer Research 51: 4505-4510) represent two particularly active areas of applied research, although the technology is also a powerful basic science research tool for the functional assessment of specific genes in cellular growth and differentiation (Simons, M. et al. (1992) Nature 359: 67-70).
While sufficient evidence indicates that oligonucleotides can cross the multiple cellular membrane barriers needed to reach their intracellular targets (Loke, S. L. et al. (1989) Proc. Natl. Acad. Sci. USA 86: 3474-3478; Yakubov, L. et al. (1989) Proc. Natl. Acad. Sci. USA 86: 6454-6458; Wu-Pong, S. et al. (1992) Pharmaceutical Research 9: 1010-1017; and Noonberg, S. B. et al. (1993) J. Invest. Dermatol. 101: 727-731), a growing number of reports suggest that this uptake process is highly inefficient and may exhibit cell-type specificity and heterogeneity (Noonberg, S. B. et al. (1993) J. Invest. Dermatol. 101: 727-731; Krieg, A. M. et al. (1991) Antisense Research and Development 1: 161-171; and Iverson, P. L. et al. (1992) Antisense Research and Development 2: 211-222). In addition, imaging studies demonstrate that the typical pattern of oligonucleotide uptake results in oligonucleotide compartmentalization within punctate vesicles believed to be of endosomal origin (Loke, S. L. et al. (1989) Proc. Natl. Acad. Sci. USA 86: 3474-3478; and Yakubov, L. et al. (1989) Proc. Natl. Acad. Sci. USA 86: 6454-6458), sequestered from their DNA or RNA targets, and subject to eventual lysosomal fusion and nuclease degradation. Rapid extracellular degradation has also been noted (Wickstrom, E. (1986) J. Biochem. Biophys. Meth. 13: 97-102). Biological activity is thought to arise from the small fraction of full-length oligonucleotide that either escapes from endosomes and rapidly accumulates in the nucleus, or enters the cytoplasm by another process and similarly accumulates intranuclearly. Oligonucleotide/nucleic acid target interactions can thus occur en route to or within the nucleus.
Current strategies for the delivery of nucleic acid for antisense gene regulation fall into 1 of 2 major classes: direct extracellular addition of short DNA oligonucleotides (or analogues thereof) to cell culture media (as described in the previous chapter), or cellular gene transfection which is transcribed by RNA polymerase II into a long antisense transcript (or mRNA with antisense insert). Strategies for the delivery of nucleic acids for triplex gene regulation fall entirely into the first class, while strategies for the delivery of nucleic acid for ribozyme gene regulation fall entirely into the second class. Both classes have clear advantages, but both are also handicapped by limitations which have yet to be adequately circumvented (Table I).
TABLE I ______________________________________ Advantages and Limitations to Current Nucleic Acid Delivery Strategies Advantages Limitations ______________________________________ Oligodeoxyribo- Easy introduction Uptake can be nucleotides to cells. inefficient, heterogeneous. Minimal Rapid intra/extracellular secondary degradation. structure concerns. No transfection Possible toxicity. required. Accommodates Noncontinuous chemical administration. analogues Can be used for High cost of synthesis antisense or triplex and purification. strategies. Polymerase II Intracellular Variable expression. transcripts expression. Can be inducible. Rapid intracellular degradation. Can be made Long transcripts with permanent. variable start and stop positions. Inexpensive. Considerable secondary structure can mask binding regions. ______________________________________
Perhaps the biggest advantage of extracellular addition of oligonucleotides translates into the biggest disadvantage of intracellular generation of polymerase II transcripts--namely the length of the nucleic acid delivered. Oligonucleotides are, by their very name, short fragments of nucleic acid, and are thus generally capable of weak or predictable secondary and/or intermolecular structures. As such, their binding regions are expected to display increased accessibility to an intracellular nucleic acid target (whose accessibility may be difficult to predict). In contrast, polymerase II transcripts are generally very long (&gt;1 kilobase) with highly variable trailing 3' sequences. These long and variable transcripts inevitably lead to complex secondary and tertiary structures which may mask key binding sequences. In most cases, a structural prediction of these long transcripts cannot be accurately determined.
Analogously, perhaps the biggest advantage of intracellular generation of polymerase II transcripts translates into the biggest disadvantage of extracellular addition of oligonucleotides--namely the site of nucleic acid delivery. Polymerase II transcripts are, by their very nature, generated intracellularly and thus do not need to cross the cell membrane barrier. For nuclear targets, the transcript is already in the correct subcellular compartment, while for cytoplasmic targets, the transcript must be actively or passively transported across the nuclear membrane. In contrast, extracellularly-added oligonucleotides must first cross the cell membrane, a process which has been shown to be inefficient, heterogeneous, cell-type specific, and to often lead to endosomal sequestration and subsequent lysosomal degradation. In addition, nucleic acid from both strategies are also susceptible to rapid degradation by cellular endonucleases and exonucleases, and both strategies may give rise to low or variable intracellular nucleic acid concentrations.
A logical question then becomes, can one design a system which combines the major advantages of both strategies while eliminating the major disadvantages? In positing such a system, one can put forth the following design criteria:
1. The system must be capable of delivering short nucleic acids of a pre-specified sequence and length to allow for adequate secondary structure prediction.
2. The system must be capable of delivering nucleic acid intracellularly.
3. The system must be capable of delivering nucleic acids of sufficient intracellular stability against nuclease degradation.
4. The system must be capable of delivering nucleic acids in high yield without cell-type specificity.